The current basic understanding of gene expression has, as its source, years of cell transfection research. Early research involved introduction of genes or other defined segments of DNA into bacteria followed by assessment of the genes' ability to function normally. More recent transfection research has centered on cell life forms higher than the bacterium, and for at least several years it has been routine to introduce cloned genes into the genomes of a variety of mammalian cells.
The transfer of genes or defined segments of DNA into cells is known by a variety of synonymous terms all of which may denote either stable or transient transfer: functional DNA uptake, DNA (or gene) transmission or transfer, or DNA (or gene) transfection. Other genetic material can also be the subject of transfection, and this is known in the art. For the purposes of the following discussion and disclosure, therefore, all of these terms should be considered interchangeable.
Transfection techniques, while they are now routine, are not always as efficient as a researcher might want or need. For example, for a long time tumor virus DNA that was free of viral coat proteins was able to induce viral multiplication and give rise to progeny virus particles when it was added to susceptible host cells, rendering them cancerous. Unfortunately, the efficiency of this technique was so low it was useless for practical research. The technique became useful for basic research when it was discovered that purified DNA from adenovirus, when precipitated with Ca.sup.++ (for example from Ca.sub.3 (PO.sub.4).sub.2) and added to a monolayer of normal rat cells growing in culture, had a higher transfection efficiency than when Ca.sup.++ was absent. The mechanism of the increased transfection efficiency is presumed to be a Ca.sup.++ -induced cellular phagocytosis of the infecting DNA particles, or PO.sub.4 crystal-induced membrane disruption allowing DNA to enter the cytoplasm.
The above-described stimulus for transfection did not solve all transfection efficiency problems, however, because only a very small fraction of the added DNA finally became functionally integrated into the cellular DNA. Introduction of Ca.sup.++ also affected cell biochemistry and metabolism, sometimes to an unacceptable degree.
Other transfection enhancing techniques are also well known. Diethylaminoethyl dextran (DEAE-dextran) can be used to bind genetic material such as DNA to generate a particulate DNA complex which enters host cells more readily than unbound DNA does. Another method uses polybrene, a detergent, which generates holes in the cell membranes to allow foreign genetic material to enter. Yet another method is called "lipofection", which is the inclusion of DNA or other genetic material into lipid vesicles which are inherently more able to coalesce with and cross the cell membrane to deliver genetic material to the cytoplasmic space than is the DNA itself. Finally, electrophoresis has also been used to "electroporate" the genetic material into the cells to achieve delivery and to enhance transfection. In the latter case, a brief, pulsed high voltage field creates membrane disruption and vectorially drives genetic material through the plasma membrane into the cytoplasmic space. Alternatively, the genetic material may enter the cell after electroporation by diffusion.
The DEAE-dextran, polybrene, lipofection, electroporation, Ca.sup.++ and other techniques all share the same general disadvantages: the ions or other substances, or the electricity, may well enhance transfection but also have greater or lesser deleterious effects on cell morphology or metabolism. Electroporation, in particular, can be fatal to cells due to generation of unwanted heat. These techniques rely on creating holes in the plasma membrane of target cells so that particulate DNA may enter by diffusion or, in the case of lipofection, so that the membranes of genetic material containing vesicles will merge with the target cell plasma membrane to achieve cellular uptake. These techniques do not stimulate phagocytosis by the target cells as a method to increase the efficiency of DNA uptake.
Accordingly, a need thus remains for a transfection enhancement method which is widely applicable and highly effective and, preferably, is biochemically and electrically neutral.